Angiogenesis is required for the growth and metastasis of cancers. One of the critical events that occur during tumor formation is the remodeling of the angiostatic vascular microenvironment to a pro-angiogenic microenvironment. This remodeling provides pro- angiogenic communications to vascular endothelial cells (ECs) that are critical for normal and pathological angiogenesis. Therefore, therapeutic interventions that disrupt communications between ECs and their microenvironment represent a potential approach for inhibiting angiogenesis and cancer. Unfortunately, there remains a great deal to learn about the microenvironment molecules that regulate angiogenesis and the molecular mechanisms by which these molecules functions. To address this problem, we recently conducted a microarray based transcriptome analysis of ECs undergoing angiogenesis in vitro. We identified 39 secreted proteins that were not previously associated with angiogenesis. Our first analysis of this data set was performed by employing a retroviral overexpression strategy in endothelial cells. Overall, this approach confirmed that two out of seven investigate proteins (i.e. MAGP-2 and lumican) control angiogenesis both in vitro and in vivo. The overall goal of the parent grant to which this revision application is amended is to characterize the molecular mechanisms by which MAGP-2 promotes angiogenesis. We have made significant progress on this project and have determined that MAGP-2 controls angiogenesis by interacting with Notch signaling pathways. Ongoing experimentation in the parent grant is dissecting the molecular details by which MAGP-2 manipulates Notch signaling and the role of endothelial specific MAGP-2 containing granules in angiogenesis regulation. While this project will help us to understand basic aspects of angiogenesis regulation, it does not seek to pursue additional regulators of angiogenesis present within the original microarray data set. This is a missed opportunity since it is highly likely that additional novel regulators of angiogenesis remain to be discovered in our original microarray data. The goal of this revision application is to discover and characterize additional stromal and cell membrane bound regulators of angiogenesis that were not investigated in the initial analysis of our microarray results. This is an important and focused project that based on prior success, has a high probability to uncover novel regulators of angiogenesis. Importantly, in accord with the R15 funding mechanism and the ARRA, the proposed projects will provide outstanding training opportunities for student researchers, will make a strong investment in an institution that has not received significant NIH funding, and therefore will help drive economic recovery in rural Indiana. We propose two specific aims to achieve our goals. In aim 1, we will identify additional stromal and cell membrane bound regulators of angiogenesis. To accomplish this, we will employ a morpholino based knockdown approach of candidate genes in zebrafish embryos to determine if these genes are important regulators of angiogenesis. The effect of gene knockdown will be monitored by microangiogram analysis of Fli1-GFP + GATA-1 RFP double transgenic zebrafish lines. This approach will allow us to rapidly and efficiently screen through the remaining putative regulators of angiogenesis in our original microarray data. Since other regulators of angiogenesis have already been isolated from this dataset, we are confident this project will uncover additional novel angiogenesis regulators. Indeed, as shown in our preliminary results, we have already identified 4 additional putative regulators of angiogenesis using this procedure. In aim 2 these new putative regulators of angiogenesis plus additional genes identified in aim 1 will be monitored for either pro- or anti-angiogenic activities. To accomplish this, candidate genes will be overexpressed and/or knocked down in mammalian endothelial cells and compared to their control counterparts for altered angiogenic activities including cell invasion, proliferation, sensitivity to angiogenic growth factors, and formation of capillary-like structures. PUBLIC HEALTH RELEVANCE: Cancer continues to be a leading cause of death and suffering in the United States. Since all solid tumors depend on the infiltration of new blood vessels (i.e. angiogenesis), strategies to block angiogenesis may provide therapeutic opportunities to treat cancer. To address this medical need, this proposal seeks to characterize new molecules and mechanisms by which angiogenesis is controlled so that we can one day use this information to block angiogenesis and combat cancer.